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| Construction and Identification of Bovine (Bos taurus) Flag and HA Epitope-tagged h3f3b Eukaryotic Expression Vector |
| 1, 1, 1,Fusheng Quan2,Yong ZHANG |
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Abstract Histone variant H3.3 is an essential maternal factor for oocyte reprogramming. After SCNT(somatic cell nuclear transfer), the H3.3 protein in oocyte will replace its counterpart in donor nucleus, opening chromatin structure and facilitating reprogramming. However, the H3.3 protein, no matter it is in oocyte or in donor cell, have same amino acid sequence. Besides, there is no specific antibody for H3.3. In order to study the replace process, we used a method of adding a tag. H3f3b is the coding gene of H3.3. Firstly, we got the CDS of h3f3b from bovine oocyte cDNA, By primer designing, we added a Flag or HA coding sequence to its 5' end. Then the sequence was connected to the eukaryotic expression vector pcDNA3.1(+).The constructed vectors of pcDNA3.1(+)-HA-h3f3b and pcDNA3.1(+)-Flag-h3f3b were identified by restriction enzyme digestion and nucleotide sequencing. After transfection of 293T cell, we confirmed the expression and location of the HA and Flag tagged H3.3 protein. Reverse transcription-PCR (RT-PCR) and Western blot showed that only the group transfected with reconstructed plasmid could transcribe reconstructed H3.3 mRNA and expressed the fusion protein. Immunofluorescent staining manifested that the protein successfully locates to nucleus. In this study, we constructed two epitope-tagged h3f3b expression vector and confirmed its expression and location, our work can be useful for the research of H3.3 exchange. Furthermore, this method can be used to study other maternal factors which have the feature of replace its somatic counterpart.
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Received: 27 March 2015
Published: 24 July 2015
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Corresponding Authors:
Fusheng Quan
E-mail: quanfusheng@nwsuaf.edu.cn
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